MicroRNAs are predicted to regulate a majority of genes in human cells and both overexpression and loss of expression of some miRNAs are correlated with malignant phenotypes in different cancer cells. A decrease in miRNA activity is most commonly observed and may be important for the plasticity of tumor cells to undergo transitions between differentiation states and to grow in different niches. In Aim 1 of this Project we plan to investigate the consequences of either partial or total loss of microRNAs in control of the stability of differentiation and quiescent states of tumor cells. Tumor cells from a mouse sarcoma cell line and from transformed mesenchymal stem cells have been isolated that are null for Dicer activity and missing miRNAs. Preliminary results show that Rb-type proteins regulate expression of miRNAs (see Project 3). The ability of these cells to undergo differentiation, survive stress and generate tumors in xenograft challenges will be explored. The plasticity of cell state due to the loss of miRNAs in the presence or absence of Rb-type proteins is being investigated in collaboration with Lees lab as outlined in Project 3. The expression of non-coding small RNAs in these Dicer null cells will also be investigated using deep sequencing technology. Preliminary results indicate that all significant miRNAs decrease by 100 fold in Dicer null cells. Aim 2 of this Project is designed to investigate the potential roles of RNAi related processes in control of transcriptional and RNA processing in the nucleus of normal and malignant cells. The activities of the RNAi-related small non-coding RNAs are probably mediated by Argonaute proteins. An objective is to define the roles of Argonaute-type proteins in the nucleus and their association with genomic sequences. Transcriptional silencing by RNAi-related small RNAs in non-vertebrate organisms is mediated by recognition of nascent RNA. In Aim 3 of this Project we will investigate the roles of long non-coding RNAs in regulation of transcription and RNA-related processes in normal and tumor cells. A majority of the promoters for genes expressed in embryonic stem cells have a second divergent polymerase paused in the antisense direction about 250 base pairs upstream of the sense polymerase. Elongation by this anti-sense polymerase is controlled by P-TEFb (Positive Transcription Elongation Factor b) and can produce RNAs thousands of nucleotides long. Establishing the roles of these and other types of long non-coding RNAs in cancer is also an objective. In particular, we will collaborate with the Jacks lab in investigating long noncoding RNAs and cancer.